Crystalline form of cyclosporine a, methods of preparation, and methods for use thereof

ABSTRACT

The present invention relates generally to crystalline forms of cyclosporine A and particularly to a newly identified form of cyclosporine A. The invention further relates to methods for its preparation and to methods for treating certain ocular disorders.

RELATED APPLICATIONS

This application is a divisional of copending U.S. patent applicationSer. No. 13/480,710, filed on May 25, 2012, which claims priority toU.S. Provisional patent application Ser. No. 61/490,887 filed on May 27,2011, the entire disclosure of both applications are incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates generally to a new crystalline form ofcyclosporine A and particularly pharmaceutical use of the newlyidentified form of cyclosporine A. The invention further relates tomethods for its preparation and to methods for treating certain oculardisorders.

BACKGROUND OF THE INVENTION

The exposed part of a normal eye is covered by a thin tear film. Thepresence of a continuous tear film is important for the well-being ofthe corneal and conjunctival epithelium and provides the cornea with anoptically high quality surface. In addition, the aqueous part of thetear film acts as a lubricant to the eyelids during blinking of thelids. Furthermore, certain enzymes contained in the tear fluid, forexample immunoglobin A, lysozyme and beta lysin, are known to havebacteriostatic properties.

A sound lacrimal system functions to form and maintain a properlystructured, continuous tear film. The lacrimal apparatus consists of thesecretory system (the source), the distribution system, and theexcretory system (the sink). In the secretory system, aqueous tears aresupplied by main and accessory lacrimal glands.

The bulk of the tear film is made of such aqueous tear. The continuousproduction and drainage of aqueous tear is important in maintaining thecorneal and conjunctival epithelium in a moist state, in providingnutrients for epithelial respiration, in supplying bacteriostatic agentsand in cleaning the ocular surface by the flushing action of tearmovement.

Surgical procedures have been suggested in the management of dry eyestates. Where there has been significant conjunctival destruction,mucous membrane transplants have been advocated. It has also beensuggested that parotid (saliva) duct transplantation can be useful inthe management of dry eyes. However, surgical alterations to combat dryeye conditions constitute a dramatic remedy and any benefit resultingfrom these alterations is questionable.

Other diseases of the eye include phacoanaphylactic endophthalmitis,uveitis, and keratoconjunctivitis sicca (KCS). These diseases can belocated throughout the eye, in both the posterior and anterior chambersof the eye as well as in the vitreous body.

Uveitis, the inflammation of the uvea, is responsible for about 10% ofthe visual impairment in the United States. Phacoanaphylacticendophthalmitis is a human autoimmune disease.

Panuveitis refers to inflammation of the entire uveal (vascular) layerof the eye. Posterior uveitis generally refers to chorioentinitis, andanterior uveitis refers to iridocyclitis. The inflammatory products(i.e. cells, fibrins, excess proteins) of these inflammations arecommonly found in the fluid spaces if the eye, i.e. anterior chamber,posterior chamber and vitreous space as well as infiltrating the tissueintimately involved in the inflammatory response. Uveitis may occurfollowing surgical or traumatic injury to the eye; as a component of anautoimmune disorder, i.e. rheumatoid arthritis, Behcet's disease,ankylosing spondylitis, sarcoidosis; as an isolated immune mediatedocular disorder, i.e. pars planitis, iridocyclitis etc., unassociatedwith known etiologies; and following certain systemic diseases whichcause antibody-antigen complexes to be deposited in the uveal tissues.Together these disorders represent the non-infectious uveitities.

Phacoanaphylaxis is a severe form of uveitis in which the lens in thecausative antigen. The lens proteins are normally secluded by the lenscapsule since before birth. When these proteins are released into theeye by injury or by surgery or occasionally during cataract development,they can become intensely antigenic and incite an autoimmune response.If the response is moderate it is seen as chronic uveitis. If it is veryfast in progression the eye becomes seriously inflamed in all segments.This latter response is named phacoanaphylaxis.

Cyclosporines are a group of nonpolar cyclic oligopeptides with knownimmunosuppressant activity. Cyclosporin A, along with several otherminor metabolites, as well as cyclosporin B, C, D, E, F, G, H, I, J, K,L, M, N, O, P, Q, R, S, T, U, V, W, X, Y and Z, have been identified.The use of cyclosporine A and cyclosporine A derivatives to treat theophthalmic conditions set forth above has been the subject of variouspatents, for example Ding et al U.S. Pat. No. 5,474,979; Garst U.S. Pat.No. 6,254,860; and Garst U.S. Pat. No. 6,350,442, the disclosure of eachof which is incorporated in its entirely herein by reference. Withrespect to its solid state chemistry, cyclosporine A (CsA) is known toexist in an amorphous form, liquid crystal form, tetragonal crystallineform (Form 1), and an orthorhombic form (Form 3).

SUMMARY OF THE INVENTION

The present invention provides a new crystalline form of CsA, withunique and novel properties suitable for pharmaceutical development.

In another embodiment of the invention, there are providedpharmaceutical compositions including a therapeutically effective amountof cyclosporine A in a new crystalline form in an ophthalmicallyacceptable carrier.

In another embodiment there are provided methods for treating an aqueousdeficient dry eye state, uveitis or phacoanaphylactic endophthalmitis inan eye. Such methods can be performed, for example, by administering toa subject in need thereof cyclosporine A in crystalline form 2 in anophthalmically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts characteristic X-ray powder diffraction (XRPD) patternsof CsA in a new crystalline form (designated as Form 2 herein),tetragonal form (designated as Form 1 herein), and orthorhombic form(designated as Form 3 herein).

FIG. 2 depicts the XRPD diffractogram of CsA crystalline Form 2.

FIG. 3 depicts the water sorption/desorption profile of CsA Form 2.

FIG. 4 depicts MDSC analysis of CsA Form 2 recovered from 0.04%formulation with 1% PS80.

FIG. 5 depicts the XRPD diffractograms for samples collected from anaqueous suspension containing 1% w/v polysorbate 80 and excess CsA Form2 after storage for 24 months.

FIG. 6 depicts the XRPD diffractograms for samples collected from anaqueous suspension containing 5% w/v hyaluronic acid and excess CsA Form2 after storage for 6 months.

FIG. 7 depicts the simulated XRPD pattern of cyclosporine A forms.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention claimed. As used herein, theuse of the singular includes the plural unless specifically statedotherwise. As used herein, “or” means “and/or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as“includes,” and “included,” is not limiting. The section headings usedherein are for organizational purposes only and are not to be construedas limiting the subject matter described.

In addition, it is to be understood that “crystalline form” and“pseudomorphic form” may be used interchangeably throughout thespecification. “Crystalline form 1” or “crystalline form 2” may also bereferred to as “Pseudomorph 1” or “Pseudomorph 2”.

Unless specific definitions are provided, the nomenclatures utilized inconnection with, and the laboratory procedures and techniques ofanalytical chemistry, synthetic organic and inorganic chemistrydescribed herein are those known in the art. Standard chemical symbolsare used interchangeably with the full names represented by suchsymbols. Thus, for example, the terms “hydrogen” and “H” are understoodto have identical meaning. Standard techniques may be used for chemicalsyntheses, chemical analyses, and formulation.

The present invention provides a new crystalline form of CsA, designatedcyclosporine A Form 2. The XRPD pattern of this novel Form 2 differssignificantly from the tetragonal form and orthorhombic form (FIG. 1).The major crystalline peaks for CsA form 2 appear at (2 θ) when scannedby an X-ray diffractometer with X-ray source as Cu Kα radiation, λ=1.54Å, at 30 kV/15 mA: 7.5, 8.8, 10.2, 11.3, 12.7, 13.8, 14.5, 15.6 and 17.5(d-spacing in crystal lattice at about 11.8, 10.0, 8.7, 7.8, 7.0, 6.4,6.1, 5.6 and 5.1 Å, respectively, FIG. 2). These major peaks are definedas those being unique to Form 2 relative to the orthorhombic ortetragonal forms; as well as, peaks having an intensity greater than 5times the background.

In one embodiment, the new crystalline form (Form 2) of CsA is anonstoichiometric hydrate of Cyclosporin A.

In another embodiment, the crystalline Form 2 is represented by theformula:

wherein X is the number of molecules of water and varies from 0-3. Inone embodiment, X in the above formula is 2.

Form 2 appears to be a kinetically stable form of CsA in aqueoussuspensions. Suspensions containing Form 2 show no conversion to otherknown polymorphic or pseudomorphic forms upon storage. It has been foundthat Form 1 and the amorphous form convert to Form 2 in the presence ofwater.

The single crystal structure of the newly discovered hydrate form ofcyclosporine A (Form 2) was determined and the crystal structureparameters are listed in Table 2. These results indicate that Form 2 isa unique compared to other known crystalline forms of cyclosporine A.

TABLE 1 Crystal data and data collection parameters of crystal structuresolution of CsA Form 2. formula C₆₂H₁₁₅N₁₁O₁₄ formula weight 1238.67space group P 2₁ 2₁ 2₁ (No. 19) a (Å) 12.6390(5) b (Å) 19.7562(6) c (Å)29.568(2) volume (Å²) 7383.8(7) Z 4 d_(calc) (g cm⁻³) 1.114 crystaldimensions (mm) 0.27 × 0.16 × 0.12 temperature (K) 150 radiation(wavelength in Å) Cu K

 (1.54164) monochromator confocal optics linear abs coef (mm⁻¹) 0.640absorption correction applied empirical

transmission factors (min, max) 0.80, 0.93 diffractometer RigakuRAPID-II h, k, l range −13 to 13 −21 to 21 −32 to 21 2θ range (deg)5.38-115.00 mosaicity (deg) 1.31 programs used SHELXTL F₀₀₀ 2704.0weighting 1/[σ²(Fo²) + (0.0845P)² + 0.0000P] where P = (Fo² + 2Fc²)/3data collected 37360 unique data 9964 R_(int) 0.077 data used inrefinement 9964 cutoff used in R-factor calculations F_(o) ² >2.0s(F_(o) ²) data with I > 2.0s(I) 6597 number of variables 834 largestshift/esd in final cycle 0.00 R(F_(o)) 0.061 R_(w)(F_(o) ²) 0.145goodness of fit 1.037 absolute structure determination Flockparameter^(b) (0.0(3))

indicates data missing or illegible when filed

The asymmetric unit of this CsA Form 2 was found to contain onecyclosporine A molecule and two water molecules. It is possible that anysmall molecule that can hydrogen bond to water could play the role ofspace filler, which would give a range of potential structures runningfrom the orthorhombic dihydrate to distorted monoclinic dihydrate. TheXRPD pattern calculated from the single-crystal structure is shown inFIG. 7 and it matches the experimental pattern shown in FIG. 2. Thesematching patterns further corroborate that Form 2 is a unique and purecrystalline form of cyclosporine A.

Without wishing to be bound by theory, thermogravimetric analysiscombined with KF titration and vapor sorption desorption analysis (VSA)suggest that CsA Form 2 is a non-stoichiometric hydrate of CsA. Thevapor sorption analysis of Cyclosporine Form 2 indicates that watercontent in the new crystal form reversibly varies with relative humidityas shown in FIG. 3. Similar to the tetragonal form, the new CsA formundergoes a phase transition to a liquid crystal or amorphous form at124.4° C. prior to melting as indicated by the modulated differentialcalorimetric (MDSC) analysis (FIG. 4).

The new physical form of CsA has a higher solubility (130 μg/mL) thanorthorhombic form (100 μg/mL)in ophthalmic formulation vehiclescontaining 1% PS80. This is desirable for developing solution orsuspension formulations. The new form appears to be a more stable formthan the tetragonal in aqueous solution. Form 2 offers some advantagesover the tetragonal and orthorhombic forms in alternative formulationssuch as ocular implants, tablets, capsules and semi-solid formulations,liquid gel capsules, suspensions and micro-emulsions.

In addition, it has been discovered that Form 2 is more readily millablethan Forms 1 or 3. Milling is very important since, in a Cyclosporin Asustained release suspension, large particles (i.e., ≧40 μm) have beenobserved to settle in a 2% hyaluronic acid (hydrogel) formulation and tobe difficult to re-suspend. The CsA Form 2 is readily milled to 10 μm orsmaller. Physically stable suspensions of these crystals have beenprepared at concentrations of up to 10% in 2.5% hyaluronic acid.

In addition to physical stability, it is anticipated that smallerparticle size will deliver more drug to the tissue, by virtue of theincreased surface area. Reducing particle size for this reason may becritical because Form 2 appears to have lower dissolutioncharacteristics than the amorphous form and therefore will likely havelower delivery to the tissue, although the smaller particle size maymitigate this problem. Indeed, it has been discovered that makingnanoparticles is easier with Form 2 than Forms 1 or 3. So, if it isrequired to nanosize the crystals in order to improve drug delivery tothe tissue and/or to improve the physical stability of the suspension,Form 2 provides a distinct advantage over other forms.

Pharmaceutical compositions may be prepared by combining atherapeutically effective amount of CsA Form 2 according to theinvention, or a pharmaceutically acceptable salt thereof, as an activeingredient, with conventional ophthalmically acceptable pharmaceuticalexcipients, and by preparation of unit dosage forms suitable for topicalocular use. The therapeutically efficient amount typically is betweenabout 0.0001 and about 5% (w/v), preferably about 0.001 to about 1.0%(w/v) in liquid formulations.

For ophthalmic application, preferably solutions are prepared using aphysiological saline solution as a major vehicle. The pH of suchophthalmic solutions should preferably be maintained between 4.5 and 8.0with an appropriate buffer system, a neutral pH being preferred but notessential. The formulations may also contain conventional,pharmaceutically acceptable preservatives, stabilizers and surfactants.

Preferred preservatives that may be used in the pharmaceuticalcompositions of the present invention include, but are not limited to,benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetateand phenylmercuric nitrate. A preferred surfactant is, for example,Tween 80. Likewise, various preferred vehicles may be used in theophthalmic preparations of the present invention. These vehiclesinclude, but are not limited to, polyvinyl alcohol, povidone,hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose,hydroxyethyl cellulose cyclodextrin and purified water.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. Accordingly, buffersinclude acetate buffers, citrate buffers, phosphate buffers and boratebuffers. Acids or bases may be used to adjust the pH of theseformulations as needed.

In a similar vein, an ophthalmically acceptable antioxidant for use inthe present invention includes, but is not limited to, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene.

Other excipient components which may be included in the ophthalmicpreparations are chelating agents. The preferred chelating agent isedetate disodium, although other chelating agents may also be used inplace of or in conjunction with it.

The ingredients are usually used in the following amounts:

Ingredient Amount (% w/w) active ingredient about 0.001-5 preservative0-0.10 vehicle 0-40 tonicity adjustor 0-10 buffer 0.01-10 pH adjustorq.s. pH 4.5-7.5 antioxidant as needed surfactant as needed purifiedwater as needed to make 100%

The actual dose of the active compounds of the present invention dependson the specific compound, and on the condition to be treated; theselection of the appropriate dose is well within the knowledge of theskilled artisan.

The pharmaceutical compositions containing CsA Form 2 are useful intreating a variety of ocular disorders. Thus, in another embodiment ofthe invention there are provided methods for treating an aqueousdeficient dry eye state, uveitis, phacoanaphylactic endophthalmitis, orkeratoconjunctivitis sicca (KCS) in an eye, comprising administering toa subject in need thereof cyclosporine A in crystalline form 2 in anophthalmically acceptable carrier.

One aspect of the present invention relates to pharmaceuticalcompositions for alleviating dry eye related symptoms, for example, asin patients having immune mediated keratoconjunctivitis sicca (KCS) ordry eye disease or other autoimmune dysfunction of the lacrimal gland,as well as dry eye symptoms of contact lens wearers.

Dry eye generally refers to any tear film abnormality, usually withepithelial abnormalities. A specific deficiency of the aqueous componentof the tear film is known as keratoconjunctivitis sicca (KCS), whichaffects about 30 million people worldwide. It is usually included aspart of Sjogren's syndrome. Literally the term denotes inflammation ofthe cornea and conjunctiva secondary to drying.

When the tear film fails to perform its functions of lubrication,oxygenation, and removal of debris, symptoms of foreign body sensation(grittiness, scratchiness, sandiness), fatigue, and dryness result. Apatient may experience severe pain, especially in the presence offilamentary keratopathy. Loss of the smooth refractive surface of thetear film causes blurred vision, which can vary from blink to blink,accounting for a variable manifest refraction and for complaints ofvariable vision throughout the day. Surface drying may produce reflextearing and the misleading complaint of excess tears. Typically,symptoms of tear deficiency are worse late in the day, with prolongeduse of the eyes (as when the patient reads or watches television), andin conditions of heat, wind, and low humidity (as on the beach or skislopes). Symptoms that are worse in the morning suggest an associatedchronic blepharitis, recurrent corneal epithelial erosion, or exposurekeratopathy. Further, symptoms include superficial punctate erosions,corneal filaments, coarse mucus plaques, and epithelial defects.

As hereinabove noted, most of these symptoms result from the unstabletear film and abnormal ocular surface that diminish the ability of theocular surface to respond to environmental challenges. Dry eye syndrome,if left untreated, can cause progressive pathological changes in theconjunctival and corneal epithelium.

The etiologies of dry eye are varied. The disease generally referred toas “dry eye” may be the result of age-related decreases in systemicandrogen support to the lacrimal gland or systemic autoimmune diseasessuch as Sjogrens Syndrome. A growing body of research suggests that dryeye is the result of an underlying cytokine and receptor-mediatedinflammatory process.

Palliative agents, such as tear replacement, tear preservation, andautonomic tear stimulation, may provide complete or partial relief ofsymptoms. However, therapeutic treatments directed at the underlyinginflammatory process may prove beneficial in correcting the underlyingdisorder.

The tear film in a normal eye consists of a thin (about 6-45 um inthickness) film composed of a mucous layer lying over the cornealepithelium and an aqueous layer covering the mucous layer andepithelium, which is in turn covered by an extremely thin (0.01-0.22 um)layer of lipid molecules.

The presence of a continuous tear film is important for the well-beingof the corneal and conjunctival epithelium and provides the cornea withan optically high quality surface. In addition, the aqueous part of thetear film acts as a lubricant to the eyelids during blinking of thelids. Furthermore, certain enzymes contained in the tear fluid, forexample, immunoglobulin A, lysozyme and beta lysin, are known to havebacteriostatic properties.

It is believed that the lipid layer is responsible for retardingevaporation of water from the eye. If the lipid layer of the tear filmis disturbed by, for example, trauma, disease, irritation of the eye orcontact lens wear, excessive evaporation of water from the eye mayoccur, leaving the surface of the eye “dry” (see e.g., Cedarstaff andTomlinson, Am. J. Optometry & Physiol. Optics, 60:167-174, 1983 [tearfilm disruption in patients with keratoconjunctivitis sicca, or “dryeye”]).

A normal lacrimal system functions to form and maintain a properlystructured, continuous tear film. The lacrimal system consists of thesecretory system (the source), the distribution system and the excretorysystem (the sink). In the secretory system, aqueous tears are suppliedby the main and accessory lacrimal glands.

Excessive evaporation of water from the tear film results in oculardiscomfort (frequently experienced by the person as dryness or tiredeyes or other less frequently reported discomfort symptoms) and mayeventually lead to physiological and pathological changes in the tissueof the eye, especially in the cornea. For contact lens wearers, suchdiscomfort is particularly acute because the loss of water from the tearfilm occurs at the interface between the tear film and the lens.Further, if the lens is a hydrogel “soft” lens, excessive evaporation ofwater from the tear film can also result in excessive evaporation ofwater from the lens.

Thus taking into account this evaporation, the continuous production anddrainage of aqueous tear is important to maintaining the corneal andconjunctival epithelium in a moist state, in providing nutrients forepithelian respiration, in supplying bacteriostatic agents and incleaning the ocular surface by the flushing action of tear movement.

In relatively mild cases, the main symptom of KCS is a foreign bodysensation or a mild “scratchiness”. This can progress to become aconstant, intense burning irritative sensation which can be debilitatingto the patient. More severe forms of KCS progress to the development offilamentary keratitis, a painful condition characterized by theappearance of numerous strands or filaments attached to the cornealsurface. Recent evidence suggests that these filaments represent breaksin the continuity of the normal corneal epithelial cells. The shearcreated by lid motion pulls these filaments, causing pain. Management ofthis stage of KCS is very difficult.

A frequent complication of KCS is secondary infection. Severalbreakdowns in the eye's normal defense mechanism seem to occur,presumably attributable to a decrease in the concentration ofantibacterial lysozyme in the aqueous tears of a patient suffering fromKCS.

Normally, aqueous-deficient dry eye states, such as, for example, KCS,are treated by supplementation of the tears with artificial tearsubstitutes. However, relief is limited by the retention time of theadministered artificial tear solution in the eye. Typically, the effectof an artificial tear solution administered to the eye dissipates withinabout thirty to forty-five minutes. The effect of such products, whilesoothing initially, does not last long enough. The patient isinconvenienced by the necessity of repeated administration of theartificial tear solution in the eye as needed to supplement the normaltears.

The following examples are intended only to illustrate the presentinvention and should in no way be construed as limiting the subjectinvention.

EXAMPLES Example 1

A 0.05 wt % CsA aqueous solution containing 1% w/v Tween 80 was preparedand stored at 65° C. The new crystalline form of cyclosporine formed byprecipitation after 24 hrs of storage.

Example 2

Cyclosporine A (30.19 g) was suspended in 900 mL of 1% w/v Tween 80 inwater at room temp. The suspension was heated to 65° C. and seeded with0.2 g of Cyclosporine A Form 2 at 52° C. The suspension was stirred for22-23 hours at 65-61° C. Precipitated solid was recovered by vacuumfiltration, washed with water, and dried under vacuum first at 40° C.,then at room temp. The yield was 30.3 g

Example 3

Aqueous suspensions containing 1 w/v % polysorbate 80 (PS80) and excessCsA Form 2 were prepared and stored at 25° C. and 40° C. Samples of thesolid residue were collected over a 24-month period and analyzed byX-ray powder diffraction. FIG. 5 shows the XRPD diffractograms forsamples collected after 24 months. Compared to the referencediffractogram of Form 2, there are no changes indicating Form 2 isphysically stable under the conditions tested.

Example 4

A suspension of cyclosporine Form 2 in 5% w/v hyaluronic acid gel inwater was prepared and stored at 25° C. Samples were collected over a 6month time period and analyzed by X-ray powder diffraction. FIG. 6 showsthe XRPD diffractograms for samples collected after 6 months. Comparedto the reference diffractogram of Form 2, there are no changesindicating Form 2 is physically stable under the conditions tested.

While this invention has been described with respect to these specificexamples, it is understood that other modifications and variations arepossible without departing from the spirit of the invention.

What is claimed is:
 1. A method for treating an aqueous deficient dryeye state, uveitis, phacoanaphylactic endophthalmitis, orkeratoconjunctivitis sicca (KCS) in an eye, comprising administering toa subject in need thereof cyclosporine A in crystalline form 2 in anophthalmically acceptable carrier.
 2. A method for treating an ocularcondition selected from the group consisting of aqueous deficient dryeye state, uveitis, phacoanaphylactic endophthalmitis, orkeratoconjunctivitis sicca in an eye, comprising administering to asubject in need thereof cyclosporine A in crystalline form 2 in anophthalmically acceptable carrier.
 3. The method of claim 2, wherein theocular condition is aqueous deficient dry eye state.
 4. The method ofclaim 2, wherein the ocular condition is uveitis.
 5. The method of claim2, wherein the ocular condition is keratoconjunctivitis sicca.